I. PATHOGNESIS OF CEREBRAL ISCHEMIA: The discovery of ET-1 and NO has greatly contributed to our understanding of the functional changes of many organs under physiological and pathological conditions (e.g., hypertension, atherosclerosis, and stroke). Previous studies focused on interactions of endocannabinoids [2-arachidonoylglycerol (2-AG) and anandamide (ANA) with the vasoconstrictor, endothelin-1 (ET-1). Both 2-AG and ANA are produced in various organs (brain,gut) and cell [monocytes, platelets, endothelial cells (EC)]; they elicit neuromodulator, cytoprotective (i.e., brain ischemia and trauma) and cardiovascular including vasodilatory effects, which are mainly mediated through cannabinoid (CB) receptors CB1, CB2 receptors. 2-AG antioxidant properties have also been implicated to ameliorate BBB injury and edema formation induced by closed head injury (CHI). This supposition was confirmed by the observed 2-AG modulation of brain injury, BBB permeability and edema formation after CHI. In addition comparable effects between 2-AG and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl(TPL), a known antioxidant nitroxide on human brain endothelial Ca2+ and cytoskeletal responses to H2O2 (ROS) where investigated in vitro. Both substances were shown to similarly modify endothelial responses mentioned above substantiating the implicated 2-AG antioxidant properties. Latest reports indicate that the vasodilatory effects of ANA in contrast to 2-AG is mediated through the vanilloid TRPV1 receptor. Untill now most of the endothelial-dependent and endothelial-dependent vascular responses have been shown to be mediated by CB1 receptors. In view of these observations, we hypothesize a possible endothelial existence of TRPV1 receptors along with CB1 and CB2 receptors and their involvement in human brain endothelial cells (HBEC) responses to endocannabinoids and related substances. These studies led to the first clear demonstration of the constitutive expression of TRPV1, CB1, and CB2, receptors (mRNA and protein)on HBEC. It also provided evidence that these receptors are functional and possibly cooperative in these cells. In addition the results demonstrated that 2-AG acts as a ligand for TRPV1 receptors as well as for cannabinoid receptors. The novel findings of colocalization and functional capacities of TRPV1, CB1, and CB2, receptors on HBEC strongly suggest that these receptors may affect the function of cerebral microvascular endothelium and contribute to the regulation of cerebral blood flow and BBB permeability. In vivo studies in progress are designated to confirm this contention. This research program has involved the investigation of the mechanisms responsible for cerebral vascular effects observed during trauma and ischemia/reperfusion (I/R) injury in tolerized and naive animals. In particular, experiments focus on the characterization of interactions involving endocannabinoids [2-arachidonoylglycerol (2-AG) and anandamide (ANA)] with the vasoconstrictor, endothelin-1 (ET-1). Recently, 2-AG antioxidant properties have been implicated to ameliorate BBB injury and edema formation induced by closed head injury (CHI). We confirmed this by demonstrating that 2-AG modulated brain injury, BBB permeability and edema formation after CHI. Furthermore, we clarified that the vasoactive properties of 2-AG are not mediated solely by CB1 receptors, but also through CB1 and vanilloid (TRPV1) receptors. This expression (constitutive) of all three (TRPV1, CB1, and CB2) receptors (mRNA and protein) on human brain endothelial cells as reported by us, was previously unknown. The former PI retired in June 2005 and for this reason, NS002933-09 SB Cerebral Ischemia: Role Of Cell Mediators In Pathogenesis And Tolerance will be terminated for next year.